Serine and cysteine proteases

By focusing the entire compound collection on the pTyr-pyridone scaffold, the researcher made use of a privileged structural element, since the pyridone was repeatedly utilized to induce /3 strand conformations in serine and cysteine protease inhibitors [157,158]. From this point of view, the potential of a modu-... 

The a-ketoamide group is a well-known electrophile for the inhibition of both serine and cysteine proteases. Introducing a pyrazole as the N-substituent of the ketoamide provides potent inhibitors of Cat K [50]. SAR exploration of the P2-P3 residues in a series of Cat K inhibitors led to the identification of the pyrrolidine... 

Inhibitor of serine and cysteine proteases plasmin, trypsin, papain and cathepsin B Inhibitor of type 1 and type 2A protein phosphatases... 

Based on their sequence homology, disulfide connectivity, and cysteine location within the sequence and chemistry of the reactive site. Pis can be assigned to distinct families, as classified by Laskowski and Kato. Kunitz-type, Bowman—Birk-type, Potato type I and type II, and squash inhibitors are members of these families shown in Table 3. For inhibitors not falling into these classifications more families have been proposed. Pis can also be classified by their target/mode of action. Plants have been found to express Pis that target serine proteinases, cysteine proteinases, aspartic proteinases, and metallo-proteinases. Serine and cysteine protease inhibitors are the best-studied PIs. ... 

As discussed above, proteases are peptide bond hydrolases and act as catalysts in this reaction. Consequently, as catalysts they also have the potential to catalyze the reverse reaction, the formation of a peptide bond. Peptide synthesis with proteases can occur via one of two routes either in an equilibrium controlled or a kinetically controlled manner 60). In the kinetically controlled process, the enzyme acts as a transferase. The protease catalyzes the transfer of an acyl group to a nucleophile. This requires an activated substrate preferably in the form of an ester and a protected P carboxyl group. This process occurs through an acyl covalent intermediate. Hence, for kineticmly controlled reactions the eii me must go through an acyl intermediate in its mechanism and thus only serine and cysteine proteases are of use. In equilibrium controlled synthesis, the enzyme serves omy to expedite the rate at which the equilibrium is reached, however, the position of the equilibrium is unaffected by the protease. 

Recently discovered antitumor monocyclic and bicyclic (3-lactam systems [40-42] are, in general, in good agreement with the phenomenon of azetidin-2-one pharmacophore of inexhaustible pharmacological potential on account of the specific ability of its numerous derivatives to inhibit not only bacterial transpeptidase, but also mammalian serin and cystein proteases [43]. As a measure of cytotoxicity, some compounds have been assayed against nine human cancer cell lines. 

Subtilisin peptidase esterase common to serine and cysteine proteases many... 

As well as complexing the substrate to the active site, many enzymes link covalently with the substrate, or a portion of it, to form an additional intermediate. Such intermediates occur in the action of enzymes as diverse as alkaline phosphatase (phosphoryl enzyme), serine and cysteine proteases (acyl enzymes), glycosidases (acylal enzymes) and aldolases. 

Sortino et al. reported the use of light irradiation on SAMs supporting the anticancer drug flutamide to study its photoreactivity and product release of nitric oxide. The authors were able to conduct studies in the absence of noxious side effects such as singlet-oxygen photosensitization and observed that nitric oxide production halted when the light was turned off [103], Another photoactive molecule, azobenzene, has recently been exploited in the SAM community to aid in studies on cell adhesion and enzyme inhibition [104], Pearson and coworkers described the use of photoisomerizable SAMs of azobenzene to conduct a series of serine and cysteine protease inhibition experiments. When light of 340-380 nm is used, the... 

M. Strajbl, J. Florian and A. Warshel, Ab initio/LD studies of chemical reactions in solution Reference free-energy surfaces for acylation reactions occurring in serine and cysteine proteases, Int. J. Quantum Chem, 77 (2000) 44—53. 

R. Mtinud and A. C. Storer. Oxyanion hde interactions in serine and cysteine proteases. BioL Chenl Hoppe-Seyler373 393 (1992). 

There are just a few studies of the use of caspase inhibitors to prevent apoptosis. Most studies concentrate on the expression of proteins of the IAP family (XIAP being the most noticeable) and the viral components p35 and CrmA (Vives et al., 2003a). CrmA, encoded by the smallpox virus, is a pseudo-substrate for serine and cysteine proteases. It inhibits caspases 1, 8, and 10 in several cell types (Sauerwald et al., 2003). p35 is a wide-spectrum caspase inhibitor encoded by baculoviruses, and it also behaves as a pseudo-substrate, inhibiting caspases 1, 3, 6, 7, 8, and 10 (Zhou et al., 1998). XIAP is the most potent member of the IAP family. It is found in the mammalian genome and is responsible for the inhibition of caspases 3, 7, and 9 (Sauerwald et al., 2002). An increased protective effect is found in CHO and HEK-293 cells expressing a XIAP mutant resistant to degradation (XIAP-BIR123) when compared with the wild-type protein (Sauerwald et al., 2002). 

Several peptidic aldehydes have been reported to be inhibitors for either y-secretase or yS-secretase or both. Common to both series are lipophilic di- and tripeptides with bulky N-terminal protection, e.g. Z-LLL-CHO (MG132), Z-YIL-CHO, and Boc-GW-CHO. The general lack of specificity of these aldehydes and their simultaneous inhibition of serine and cysteine proteases makes interpretation of data rather cumbersome. Indirect mechanisms through general protease inhibition result in complex concentration activity observations. Z-LLL-CHO (MG132), in fact, blocks maturation of the amyloid precursor protein. Some of these drawbacks were avoided by difluoro ketones as pioneered by Merryl Dow (Scheme 3.5.6), which... 

This clearly resembles the inhibition mode of the serine and cysteine protease inhibitors described above. Iterative refinement (Figure 1.18), e.g., by variation of ring size and symmetrization of the functional decoration pattern, combined with subsequent extrapolation of the renin-specific finding to the entire aspartate protease family, is the apparent basis for a new generation of nonpeptide, lead-like inhibitors with multiple therapeutically relevant endpoint opportunities. The five-membered 3,4-di(aminomethyl)-pyrrolidine (Figure 1.18) served as the core structure for highly active aspartate protease inhibitors [101]. 

Casillas, R., Kam, C.M., Powers, J.C. (2000a). Serine and cysteine proteases in sulfur mustard-exposed hairless mouse skin enzymatic activity and inhibition profiles. J. Toxicol. Cutan. Ocul. Toxicol. 19 137-51. 

Figure 8 Irreversible inhibitors of proteases. Serine and cysteine proteases can be acylated by aza-peptides, which release an alcohol, but cannot be deacylated due to the relative unreactivity of the (thio) acyl-enzyme intermediate. Reactive carbons, such as the epoxide of E64, can alkylate the thiol of cysteine proteases. Phosphonate inhibitors form covalent bonds with the active site serine of serine proteases. Phosphonates are specific for serine proteases as a result of the rigid and well-defined oxyanion hole of the protease, which can stabilize the resulting negative charge. Mechanism-based inhibitors make two covalent bonds with their target protease. The cephalosporin above inhibits elastase [23]. After an initial acylation event that opens the p-lactam ring, there are a number of isomerization steps that eventually lead to a Michael addition to His57. Therefore, even if the serine is deacylated, the enzyme is completely inactive.

In contrast to the equilibrium-controlled approach which ends with a true equUibrium, in the protease-catalyzed kinetically controlled synthesisf l the product appearing with the highest rate and disappearing with the lowest velocity would accumulate. This approach requires the use of acyl donor esters as carboxy components (Ac-X) and is limited to proteases which rapidly form an acyl-enzyme intermediate (Ac-E). Serine and cysteine proteases are known to catalyze acyl transfer from specific substrates to various nucleophihc amino components via an acyl-enzyme intermediate. In reactions of this type, the protease reacts rapidly with an amino acid or peptide ester, Ac-X, to form a covalent acyl-enzyme intermediate, Ac-E, that reacts, in competition with water, with the amino acid or peptide-derived nucleophile HN to form a new peptide bond (Scheme 3). The partitioning of the acyl-enzyme intermediate between water and the added nucleophile is the rate-limiting step. Under kinetic control, and if k4[HN] k3[H20], the peptide product Ac-N should accumulate. However, the soluble peptide product will be degraded if the reaction is not terminated after the acyl donor ester is consumed. 

A step-by-step peptide synthesis from the N- to the C-terminus is not possible with chemical methods as it risks partial epimerization due to the repeated carboxy activation procedures, In constrast, the stereo- and regiospecificity of serine and cysteine proteases ensures integrity of the stereogenic center and allows ecological reaction conditions without side-chain protection. Scheme 4 shows the synthesis scheme using clostripain and chymo-trypsin as catalysts.The second coupling reaction was carried out by enzyme catalysis in a frozen aqueous system (see Section 4.2.3.1). 

The kinetic approach can be more efficiently manipulated than the thermodynamic approach, but serine and cysteine proteases are not perfect acyl transferases. Undesired reactions may take place parallel to acyl transfer, for instance hydrolysis of the acyl-enzyme, secondary hydrolysis of the formed peptide bond, and other undesired proteolytic cleavages of possible protease-labile bonds in reactants and product. The elimination or minimization of these disadvantages can be performed by various manipulations on the level of the reaction medium, the enzyme, and the substrate, as well as on mechanistic features of the process. 

Many of the known inhibitors of serine and cysteine proteases feature the same types of war-head (e.g. oc-ketoamide, 8-lactam, aldehyde) which are able to undergo a covalent interaction with the nucleophilic active site alcohol or thiol group [371-373]. Depending on the reactivity of the war-head this approach has led to irreversible inhibitors or reversible but tight-binding inhibitors. The problem with this approach is that it is difficult to achieve selectivity and so there is considerable interest in the discovery of inhibitors that bind in a non-covalent fashion to the active site. 

Proteases may be promising targets for drug intervention since proteolytic events have been shown to be critical for successful invasion of the red cell by the merozoite (Blackman, 2000 O Donnell and Blackman, 2005). For example, in vitro invasion by falciparum merozoites can be blocked by the serine and cysteine protease inhibitors, chymostatin and leupeptin moreover, these inhibitors act at different steps in the invasion process. Chymostatin inhibited both invasion and rupture of... 

In view of the apparent convergent evolution of mechanism in the serine and cysteine protease family, it is interesting that two phosphodiesterases that require Ca + for catalytic activity by virtue of presumed electrophilic catalysis via direct coordination to the anionic phosphoryl oxygens of the substrate have evolved conceptually similar (general basic catalysis) but structurally distinct solutions to the problem of phosphodiester hydrolysis. 

Basic to protease catalyzed oligopeptide synthesis is equilibrium- or thermodynamic control to direct reversal of proteolysis . Difficulties encountered include low reaction rates, high stoichiometric amounts of enzyme, and the need to apply direct approaches to shift the reactions towards formation of desired products. Reaction conditions that lead to product precipitation or extraction increase efficiency of the reverse reaction. Kinetically controlled syntheses has proved useful for serine and cysteine proteases that form activated acyl enzyme intermediates during catalysis. This approach generally involves use of activated acyl moieties, such as esters, as donor components which significantly accelerate the reaction rate. This study makes use of principles from both kinetic and thermodynamically controlled reactions in that, reactants are activated by formation of esters and products precipitate fi om reactions. 

Azapeptides, -NH-CHRi-CO-NH-NR -CO-NH-CHR -CO-, a dass of backbone-modified peptides in which the a-CH of one or more amino add residues in the peptide chain is isoelectronicaUy replaced by a trivalent nitrogen atom. This alteration results in a loss of asymmetry associated with the a-CH, and yields a structure that can be considered intermediate in configuration between d- and t-amino acids. This a-carbon replacement is connected with the capability to provide resistance to enzymatic deavage, and the capacity to act as selective inhibitor of serine and cysteine proteases [J. Gante, Synthesis 1989, 405 J. Magrathetal.,J. Med. Chem. 1992,35,4279 R. Xing et al., J. Med. Chem. 1998, 42,1344 E. Wieczerzak et al., J. Med. Chem. 2002, 45, 4202]. 

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